G protein-coupled receptors (GPCR) play a critical role in almost every aspect of human physiology and disease. The protein arrestin plays a vital role in many of these pathways. Arrestin binds to the phosphorylated form of GPCRs and prevents the receptor from binding to and activating G proteins, resulting in desensitization. In addition, for many GPCRs, arrestin functions as an adapter molecule for internalization as well as "secondary" signaling. Arrestin mediates internalization via a domain that binds directly to clathrin and the adapter AP-2. Arrestins also bind to kinases and other signaling proteins. Our recent results have identified a specific molecular interaction between arrestin and AP-2 that is critical for the recycling certain GPCRs. We have also shown that disrupting this interaction induces a rapid apoptotic pathway for many GPCRs. To date there are no small molecules known that regulate interactions between arrestin and its interacting proteins. We have developed a high throughput flow cytometric assay involving AP-2 and a fluorescent arrestin peptide. The primary goal of the proposed research is to identify compounds that regulate the interaction between arrestin and AP-2, resulting in modulation of GPCR trafficking, signaling and apoptosis. A small molecule that inhibits this interaction will be a valuable tool to assess the role of AP-2 in the arrestin-dependent regulation of potentially hundreds of GPCRs and could represent the basis for therapeutic development through its ability to induce apoptosis in disease cells. PUBLIC HEALTH RELEVANCE: Receptors play a vital role in almost every aspect of human biology and disease. Receptor activity and function are mediated by a large collection of interacting proteins. The largest class of receptors is the G protein-coupled receptor (GPCR) family. The protein arrestin plays a critical role in both inhibiting and activating signaling by GPCRs. We have identified a specific interaction between arrestin and the cellular protein AP-2. To date there are no small molecules known that regulate interactions between arrestin and its interacting proteins. The goal of the proposed research is to identify compounds that inhibit or stimulate the interaction between arrestin and AP-2 to provide a valuable tool to assess the role of this interaction in the regulation of hundreds of GPCRs. Such a molecule would be of great benefit for researchers in the field and could find therapeutic utility in its ability to induce apoptosis in hyper stimulated disease cells such as cancer cells.